Numerous studies indicate that inactivation of the p53 pathway is a pivotal event in tumorigenesis of all kinds of human cancer, including breast cancer. Indeed, germ line p53 mutations were observed in the patients with familial breast cancer such as the Li-Fraumeni syndrome while between 15 to 30% of the Sporadic breast tumors have also been shown to contain somatic p53 mutations, further supporting the notion that abrogation of p53 function is critically involved in breast cancer tumorigenesis. Importantly, accumulating evidence also indicates that, in the cells that retain wild-type p53, other defects in the p53 pathway also play an important role in tumorigenesis, p53 is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis. Stabilization of p53 in response to various types of stress is essential for its tumor suppressor function. However, the molecular mechanism by which p53 is stabilized is not completely understood. We recently have identified HAUSP (a cellular deubiquitination enzyme) as a novel key factor in the p53 regulatory pathway. We have found that HAUSP specifically binds and stabilizes p53 in cells. Significantly, HAUSP has an intrinsic deubiquitination activity, and this enzymatic activity is required for HAUSP-mediated p53 stabilization. The overall objective of this application is to demonstrate the precise role of HAUSP in the regulation of p53 function, and elucidate this novel p53 regulatory pathway in tumorigenesis of breast cancer. The first specific aim is to elucidate the role for HAUSP in the regulation of p53 function in breast cancer cell lines. We will examine the in vivo interactions between p53 and HAUSP in breast cancer cell lines and also test the role of HAUSP in p53-dependent cell growth arrest and apoptosis in these cells. The second specific aim is to define the physiological role of HAUSP in tumorigenesis of breast cancer. We will develop a mouse model for a conditional knockout of m-HAUSP in breast tissues to assess for breast tumor formation and developmental phenotypes. The third aim is to examine the expression levels and gene alterations of HAUSP in human breast tumors. Since HAUSP can stabilize and activate p53-mediated tumor suppressor function, HAUSP itself is a putative tumor suppressor. Thus, we will test the expression levels of HAUSP in tumor samples by immunohistochemistry, and also search for gene alterations and nmtations of HAUSP in breast tumors.